Delacroix Laurence, Begon Dominique, Chatel Guillaume, Jackers Pascale, Winkler Rosita
Molecular Oncology Laboratory, GIGA/Experimental Cancer Research Centre, University of Liege, Belgium.
DNA Cell Biol. 2005 Sep;24(9):582-94. doi: 10.1089/dna.2005.24.582.
Overexpression of the ERBB2 gene occurs in 30% of human breast cancers and is correlated with poor prognosis. The deregulation is the consequence of an increased transcription level and gene amplification. Several laboratories, including our own, have identified, in the proximal promoter, enhancers implicated in the gene overexpression. However, our previous studies of a 6-kb ERBB2 promoter fragment revealed the presence of repressing fragments, which were able to overcome the effect of the proximal enhancers. These repressing elements were functional in all cell lines, regardless of their endogenous ERBB2 expression level. Here, we show that a distal ERBB2 promoter region restores high transcription rates specifically in ERBB2 overexpressing breast cancer cells. This distal promoter region thus contains enhancers essential for the overexpression of the gene. By EMSA, performed with nuclear extract of cells overexpressing (BT-474) or not (MDA-MB-231) the ERBB2 gene, we show that at least two sequences of the distal promoter region are bound exclusively by BT-474 extract. Further experiments reveal that AP-2 transcription factors contribute to this differential binding activity, by binding recognition sequences located 4500 bp and 4000 bp upstream of the transcription start site. These sites are occupied by AP2 in vivo, as demonstrated by ChIP assay. Inactivation of AP-2 proteins in ERBB2 overexpressing cells reduces the distal promoter activity up to 70%, indicating the AP-2 factors are implicated in the strong distal enhancing effect. Moreover, we identified a 54-bp fragment that is bound specifically by BT-474 nuclear extract. Further experiments did not lead to the identification of the protein responsible for this binding. Our results thus highlight the importance of ERBB2 distal promoter region and further implicate AP-2 in ERBB2 overexpression in breast cancer cells.
ERBB2基因在30%的人类乳腺癌中过表达，且与预后不良相关。这种失调是转录水平增加和基因扩增的结果。包括我们自己实验室在内的几个实验室，已在近端启动子中鉴定出与该基因过表达相关的增强子。然而，我们之前对一个6 kb的ERBB2启动子片段的研究发现了存在抑制片段，这些片段能够克服近端增强子的作用。这些抑制元件在所有细胞系中均有功能，无论其内源ERBB2表达水平如何。在这里，我们表明ERBB2启动子远端区域特异性地在ERBB2过表达的乳腺癌细胞中恢复了高转录率。因此，这个远端启动子区域包含该基因过表达所必需的增强子。通过用ERBB2基因过表达（BT - 474）或未过表达（MDA - MB - 231）的细胞的核提取物进行电泳迁移率变动分析（EMSA），我们表明远端启动子区域的至少两个序列仅被BT - 474提取物结合。进一步的实验表明，AP - 2转录因子通过结合位于转录起始位点上游4500 bp和4000 bp处的识别序列，促成了这种差异结合活性。如染色质免疫沉淀分析（ChIP）所示，这些位点在体内被AP2占据。在ERBB2过表达细胞中使AP - 2蛋白失活可将远端启动子活性降低多达70%，表明AP - 2因子参与了强大的远端增强作用。此外，我们鉴定出一个54 bp的片段，它被BT - 474核提取物特异性结合。进一步的实验未能鉴定出负责这种结合的蛋白质。因此，我们的结果突出了ERBB2启动子远端区域的重要性，并进一步表明AP - 2与乳腺癌细胞中ERBB2的过表达有关。
